Project Summary/Abstract Autism is a neurodevelopmental disorder that affects 1 in 68 children and is characterized by lifelong impairment in social interactions. Autism affects multiple brain structures including the prefrontal cortex, but the underlying molecular mechanisms are unknown. Recent gene sequencing studies have identi?ed hundreds of mutations associated with the disease. Since each mutation is individually rare, studying a single mutation does not provide broad insight into common disease mechanisms. The study of many genes in parallel could provide generalized insight, but such efforts are limited to resource intensive settings such as large-scale consortia. Therefore, experimental approaches that allow a single investigator to study the functional impact for many autism mutations simultaneously would greatly expedite discovery. Human pluripotent stem cells (hPSCs) are an ideal system for parallel studies of gene function: 1) hPSCs are amenable to facile genome editing technologies, allowing rapid construction of many genetic disease lines; 2) hPSCs can differentiate into any cell-type of the body, offering unprecedented access to disease-tissue; 3) individual hPSC lines can in theory be pooled into a single assay, supporting increased throughput of genetic analysis. The goal of this study is to develop a multiplex hPSC platform where multiple disease lines are pooled into a single differentiation and analysis assay. The utility of this approach will be validated by testing a set of autism mutations for shared molecular and developmental phenotypes. Successful completion of this study will provide novel insights into underlying mechanisms of autism. Importantly, it will establish a scalable, ?exible hPSC platform that to could be applied to decode the functional architecture of all autism mutations. This proposal effectively supports the development of an aspiring physician-scientist in using human stem cells to study brain development and complex disease.